Retinoblastoma is the prototype genetic cancer caused by mutations disrupting the RB1 tumor suppressor gene. Following loss of RB1, retinoblastoma acquires further genetic changes in a characteristic set of oncogenes and tumor suppressors including gains of the oncogenes KIF14, DEK, E2F3, and MYCN and loss of the tumor suppressor CDH11. The constellation of genetic changes is the postulated genetic pathway leading to retinoblastoma. However, advances in molecular diagnostic testing for RB1 gene mutations allows detection of at least one RB1 mutation in 98% of unilateral retinoblastomas leaving 2% of cases with undetectable RB1 mutations (RB1+/+ retinoblastoma). RB1+/+ retinoblastomas have high-level MYCN gene amplification (>30 copies) and few other genetic changes. In addition, RB1+/+ retinoblastoma present earlier than conventional RB1-/- retinoblastoma and show histologic features similar to MYCN-amplified neuroblastoma. Altogether, this study describes a distinct genetic subset of retinoblastoma characterized by wild-type RB1 gene and high-level MYCN gene amplification.

Retinoblastoma is the prototype genetic cancer caused by mutations disrupting the RB1 tumor suppressor gene. Following loss of RB1, retinoblastoma acquires further genetic changes in a characteristic set of oncogenes and tumor suppressors including gains of the oncogenes KIF14, DEK, E2F3, and MYCN and loss of the tumor suppressor CDH11. The constellation of genetic changes is the postulated genetic pathway leading to retinoblastoma. However, advances in molecular diagnostic testing for RB1 gene mutations allows detection of at least one RB1 mutation in 98% of unilateral retinoblastomas leaving 2% of cases with undetectable RB1 mutations (RB1+/+ retinoblastoma). RB1+/+ retinoblastomas have high-level MYCN gene amplification (>30 copies) and few other genetic changes. In addition, RB1+/+ retinoblastoma present earlier than conventional RB1-/- retinoblastoma and show histologic features similar to MYCN-amplified neuroblastoma. Altogether, this study describes a distinct genetic subset of retinoblastoma characterized by wild-type RB1 gene and high-level MYCN gene amplification.

Neuroblastoma is the most common pediatric cancer in infants, arising from the sympathoadrenal lineage of the neural crest. Despite recent advances in other pediatric cancers, long term survival in high risk cases of neuroblastoma remains below 40%. Therefore, to develop successful therapeutics targeting high risk tumors, further research into the mechanisms involved in high risk tumor formation is necessary. Prognosis in neuroblastoma is determined by a number of factors, including certain genetic and biological variables. The genetic variable correlated most with high risk disease is amplification of the MYCN gene, which is present in ~25% of tumors. Additionally, ~70% of these MYCN-amplified tumors express the neurotrophin receptor TrkB, and its ligand, brain-derived neurotrophic factor (BDNF), with concurrent expression of these proteins correlated with high risk disease independent of MYCN-amplification status. To better understand factors influencing MYCN-amplified tumor cell phenotype, and the role of TrkB signaling in high risk neuroblastoma, the experiments in this dissertation examined growth factor effects on MYCN-amplified tumor cells from the TH-MYCN mouse model of neuroblastoma, as well as the creation, and expression of a constitutively active TrkB receptor in a neural crest derived cell line. Overexpression of MYCN targeted to the sympathoadrenal lineage by the tyrosine hydroxylase (TH) promoter is sufficient to cause neuroblastoma in 100% of mice homozygous for the transgene (TH-MYCN mice). Screening growth factors, in vitro treatment of tumor cells from dissociated TH-MYCN tumors with ciliary neurotrophic factor (CNTF) was found to promote differentiation marked by increased neurite outgrowth, and withdrawal of actively dividing cells from the cell cycle. These effects were both concentration dependent, and specific to CNTF, as all other neurotrophic factors tested had no effect on differentiation. Furthermore, TH-MYCN tumor cells were found to highly express the receptor for CNTF, CNTFR both in vitro and in vivo. Testing the ability of CNTF to affect tumor growth in vivo, CNTF treatment attenuated subcutaneous tumor growth of the TH-MYCN tumor-derived cell line NHO1S in wild type 129/SvJ mice. Therefore, CNTF signaling may be a potential therapeutic target in MYCN-amplified neuroblastoma. In addition to being significantly correlated with a poor prognosis in neuroblastoma, the presence of activated TrkB signaling promotes a more aggressive phenotype in established neuroblastoma cell lines. However, whether TrkB signaling is sufficient to transform neural crest derived cells had not been established. To determine the role of TrkB signaling in malignant transformation, the two immunoglobulin-like (Ig) ligand binding domains were removed from a full length rat TrkB receptor. Expression of this receptor, termed IgTrkB, leads to elevated phosphorylated Erk levels in the absence of ligand, indicating the receptor is constitutively active. When expressed in the neural crest derived cell line NCM-1, constitutive TrkB signaling confers a highly transformed phenotype characterized by enhanced proliferation, anchorage-independent cell growth, anoikis resistance, and matrix invasion. Furthermore, IgTrkB NCM-1 cells upregulate transcripts for a number of cancer promoting genes, in addition to the poor prognosis marker MYCN. In vivo, IgTrkB NCM-1 cells form highly aggressive tumors, requiring euthanasia of mice by 15 days following injection, while wild type cells fail to grow. Thus, TrkB signaling is sufficient to transform cells derived from the neural crest.

Medulloblastoma (MB) is the most common malignant brain tumor affecting children. The transcription factors MYCN and SOX9 are associated with initiation, maintenance and recurrence of MB and are also connected to more aggressive tumors. In this study, a ChIP was performed to isolate DNA from genes that are transcriptionally regulated by these proteins. Identification of these target genes will reveal new potential drug targets and help us better understand the functions of MYCN and SOX9. The ChIP was not fully optimized during this project and the target genes were not sent for sequencing and identified. To study the connection between SOX9 and recurrence, cells with different levels of SOX9 were treated with drugs, after which cell viability was measured. No significant difference in resistance could be measured. Change in expression level of MYCN, SOX9 and other relevant genes after drug treatment was also studied. The results show an increase in SOX9 and HES1, suggesting that these genes are involved in tumor recurrence.

Das Neuroblastom ist der dritthäufigste maligne Tumor im Kindesalter und ist für 15% der Todesfälle durch Krebs bei Kindern unter 14 Jahren verantwortlich. Viele molekularbiologische Vorgänge, die zu der heterogenen Prognose der Patienten beitragen, sind noch nicht verstanden. Als Hauptrisikomerkmal stellt sich die Amplifikation und erhöhte Expression von MYCN dar. In Vorarbeiten der Arbeitsgruppe von Prof. Christiansen zeigte MYCN Einfluss auf die Genregion von Thyroid Cancer 1 (TC1), das als neuer Marker für maligne Schilddrüsenkarzinome erkannt wurde. In der vorliegenden Arbeit wurden erste Untersuchungen zur prognostischen Bedeutung von TC1 im Neuroblastom, sowie die Charakterisierung eines TC1 Überexpressionsphänotyps humaner Neuroblastomzelllinien unter Einfluss von MYCN durchgeführt. Es wurde ein Überexpressionsvektor von TC1 in die Neuroblastomzelllinie SH-EP eingebracht, welche über ein aktivierbares MYCN- Konstrukt verfügt. Dieser neue Phänotyp wurde bezüglich der Proliferation, des Zellzyklus und der Apoptose im Vergleich zu einer Kontrollzelllinie ohne Überexpression untersucht. Eine In-silico Recherche in der Versteeg Neuroblastomdatenbank ergab eine deutlich bessere Überlebenswahrscheinlichkeit für Patientin mit hoher TC1 Expression. Es konnte gezeigt werden, dass MYCN Amplifikation und Expression in einem Panel von Neuroblastom Zelllinien nicht mit der TC1 Expression korrelieren. Die spezifische Aktivierung von MYCN führte hingegen zu einer Expressionssteigerung von TC1. Weiterhin zeigte sich, dass eine TC1 Überexpression die Proliferation hemmt, indem es die MYCN induzierte G1- S- Phasen- Transition inhibiert. TC1 zeigt antiproliferative Eigenschaften im Zellkulturmodell und stellt sich als neuer prognostisch günstiger Parameter im Neuroblastom dar.

Die Abschätzung der Prognose für Patienten, insbesondere Kinder mit onkologischen Erkrankungen stellt eine große Herausforderung an die behandelnden Ärzte dar. Vor Beginn einer Therapie werden daher viele Informationen gesammelt, um einen Patienten möglichst gut in eine vordefinierte Risikogruppe stratifizieren und dementsprechend eine mehr oder weniger intensive Therapie anbieten zu können. Diese Einteilungen sind allerdings für keinen Malignomtyp mit 100%-iger Sicherheit möglich. Das ist die Ursache dafür, dass auch in niedrige Risikogruppen eingeteilte Patienten nicht auf die Therapie ansprechen und einen unvorhergesehen schlechten Verlauf zeigen können. Auf der anderen Seite scheint es Patienten zu geben, die trotz initial schlecht eingeschätzter Prognose einen überaschend guten Verlauf nehmen, auf die Therapie gut ansprechen und letztlich geheilt werden können. Einen Beitrag zu leisten, um die Stratifizierung für Kinder, die an einem Neuroblastom erkrankt sind, zu verbessern und damit zu vermeiden, dass einige Patienten unter- oder andere Patienten übertherapiert werden müssen, ist das Ziel dieser Habilitationsarbeit. Zu diesem Zweck wurden differentielle, molekulare Marker in primären humanen Neuroblastomen identifiziert und deren prognostische Bedeutung dargestellt. Einzelne dieser Marker (differentiell expremierte mRNAs) wurden in Zellkultursystemen funktionell untersucht, um deren zellbiologische Funktion, die der jeweiligen prognostischen Bedeutung zugrunde liegen kann zu erklären. Desweiteren konnten genomische Merkmale des amplifizierten genomischen Abschnittes auf Chromosom 2p25 um MYCN beschrieben werden. Darauf basierend konnte eine patientenindividuelle und tumorzellspezifische PCR entwickelt werden (AFS-PCR), die sich als Marker für den Nachweis einer minimalen Resterkrankung eignet.

Made available in DSpace on 2015-07-22T20:50:35Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-04-28 / Meduloblastoma (MB) é o tumor maligno de sistema nervoso central (SNC) mais comum em criança, compreendendo 20% dos tumores primários de SNC e 40% dos tumores cerebelares da infância. Devido sua forte tendência metastática, o tratamento padrão pós-operatório inclui radio e quimioterapia, cujo impacto causa distúrbios endócrinos e de crescimento, e disfunção neurocognitiva a longo prazo. Frente a esses efeitos negativos, muitas pesquisas em meduloblastoma têm sido realizadas com intuito de obter conhecimento biológico desses tumores para tentar identificar fatores prognósticos moleculares que possam orientar os tratamentos, tornando-os mais específicos e menos agressivos. Alguns estudos em MB têm sugerido que a expressão do oncogene MYC está associada com diminuição da sobrevida e sua superexpressão com maior agressividade do tumor. Por isso, MYC pode ser um indicador importante de prognóstico, além de modulador do comportamento desta doença. Enquanto o gene MYC é expresso em uma variedade de tecidos, a expressão de MYCN, outro membro da família MYC, é restrita a estágios precoces do desenvolvimento embrionário de alguns tecidos apenas, entre eles, o sistema nervoso central e periférico, sendo um mediador importante dos efeitos de ativação na proliferação de células precursoras cerebelares. Dessa forma, quando a expressão de MYCN está desregulada, ela aumenta a tumorigenicidade dessas células podendo dar origem ao MB. Além disso, o gene MYC também é considerado importante regulador da transcrição TERT, gene que codifica uma subunidade catálica de da telomerase, enzima importante para carcinogênese e imortalização de células neoplásicas. A atividade anormal da telomerase está presente em 90% dos cânceres e o aumento de sua atividade está associado a eventos clínicos desfavoráveis. Outro gene importante é o ASPM (abnormal spindle-like microcephaly associated) que desempenha função fundamental na neurogênese e proliferação celular durante o desenvolvimento cerebral. Esse gene codifica uma proteína de centrossomo e fuso mitótico que permite a divisão celular simétrica em células neuroepiteliais durante o desenvolvimento e aumento do tamanho cerebral. Alterações em ASPM é a causa mais comum de microcefalia primária em humanos e de falha de segregação, induzindo a aneuploidias e instabilidade genética. Além desses genes, outro gene estudado recentemente, como alvo em xv imunoterapia, é o gene PRAME que codifica um antígeno tumoral que está presente em vários tumores, incluindo meduloblastoma. O gene PRAME possui baixa ou ausência de expressão em tecidos normais, por isso é pode ser um forte candidato como alvo em imunoterapia, que é um tratamento menos tóxico. OBJETIVOS: O objetivo desse estudo foi investigar a expressão dos genes MYC, MYCN, TERT, ASPM e PRAME em fragmentos tumorais de meduloblastoma de crianças e tentar correlacionar com os parâmetros clínicos e verificar se há correlação de MYC, MYCN, TERT entre si, uma vez que estão correlacionados. MÉTODOS: Análise de expressão gênica foi realizada através de PCR quantitativa em tempo real, utilizando sistema SYBR Green, em 37 amostras tumorais de crianças, com média de idade de 8 anos. Para comparação de perfil de expressão foi usada duas amostra de cérebro normal. A análise estatística foi realizada nos programas Graph Pad Prism 4 e VassarStats RESULTADOS: Todas nossas amostras superexpressaram o gene MYCN com valor de quantificação relativa (RQ) mediana igual a 31 com p=0.001; assim como, todas nossas amostras também superexpressaram o gene ASPM com mediana igual a 586, p<0.0001. Do total de amostras, 95%, 81% e 84% superexpressaram TERT, MYC e PRAME respectivamente, sendo os valores de RQ (mediana) iguais a 322, p=0.01; 9.2, p<0.0001; 33, p<0.0001. Apesar da elevada expressão dos genes estudados na maioria das amostras estudadas, houve apenas correlação estatística entre a superexpressão de MYCN (p=0.008) e os pacientes que foram a óbito, e de TERT e os pacientes que recidivaram (p=0.0431). Não encontramos outra correlação estatística entre a superexpressão dos genes e as características clínicas dos pacientes. CONCLUSÃO: Os genes MYC, MYCN e TERT estavam superexpressos nas amostras de meduloblastoma analisadas em uma freqüência muito superior ao demonstrado na literatura, o que sugere que esses três genes podem ajudar na identificação de tumores agressivos, uma vez que o pognóstico desses pacientes continua baseado apenas em parâmentros clínicos. A superexpressão de ASPM em todas as amostras estudadas sugere que este gene pode estar envolvido na origem de MB, como parte da neurogênse anormal durante o desenvolvimento embrionário, porém estudoas funcionais devem ser realizados para confirmar essa hipótese. Por fim, o gene PRAME pode ser candidato à marcador de célula tumoral em MB, podendo no futuro ser candidato como alvo em imunoterapias. / To investigate the expression of genes MYC, MYCN and TERT in tumor fragments of pediatric medulloblastoma and correlate gene expression profiles with clinical parameters. Analysis of gene expression was performed by quantitative PCR real time in 37 tumor samples and correlated with clinical and pathological data. All 37 samples overexpressed MYCN gene (p= 0.001), 95% and 84% of the samples overexpressed TERT and MYC, respectively (p<0.0001). Twenty nine (78%) of all samples had concomitant high expression of MYC, MYCN and TERT genes together. Seventeen (59%) were high-risk classification, 10 (34%) were metastatic (M+) stage, two (7%) were anaplastic or largecell/ anaplastic subtype, eight (28%) of patients relapsed, beyond thirteen (45%) suffered partial surgical resection. and fourteen (48%) died. We found correlation between MYC, MYCN and TERT expression (p<0.0001). The identification of a subgroup with concomitant overexpression of the three investigated genes suggests the possibility of using more than one aspect of molecular indicative of unfavorable prognosis that characterizes the group with poor outcome. However, in future this may be enhanced by targeted therapy for the product TERT as proposed in some neoplasms. The identification of molecular events in the medulloblastoma categorization aims to help at-risk groups moving towards individualized medicine. / TEDE / BV UNIFESP: Teses e dissertações

Medulloblastoma (MB) is the most common malignant pediatric brain tumor. Overall survival is about 70% and in cases where current treatment fails, the disease recurs and most often is fatal. At the molecular level, MB can be divided into four defined subgroups: WNT, SHH, Group 3 and Group 4. Amplification of MYC family genes is common in MB and correlates with poor prognosis and tumor relapse. In this thesis we showed how MYCN initiates brain tumors when transduced in neural stem cells (NSCs). Prior to transduction, NSCs were isolated from different brain regions and at various time points. While overexpression of wild-type MYCN did not generate any tumors, orthotopic transplantation of MYCNT58A-expressing forebrain, brain stem and cerebellar NSCs induced diffuse malignant glioma, PNET-like tumors and MB, respectively. Interestingly, MYCNT58A-expressing cerebellar NSCs induced SHH-dependent MB from embryonic cells but SHH-independent MB from postnatal cells. We further showed that cerebellar NSCs transduced with both MYCNT58A and transcription factor SOX9 developed tumors faster and promoted distant migration into the forebrain. The function and regulation of SOX9 in MB cells is poorly understood. We identified SOX9 protein as target of FBW7 ubiquitin ligase and demonstrated the effects of SOX9 on MB cells migration, metastasis and drug resistance. We further blocked PI3K pathway to destabilize SOX9 which sensitized cells to cytostatic treatment. We used a (TetOFF) transgenic mouse model of MYCN-induced MB (GTML) and crossed it with a (TetON) transgene which allowed us to specifically target rare SOX9-positive cells in the tumor. In this system, MB develops spontaneously and SOX9-negative tumor cells can be killed off by doxycycline. The few remaining SOX9-positive cancer cells were able to promote distant MB recurrences. Such a pattern of relapse was recently shown for Group 3 and 4 human MB where about 90% of the recurrences were distant. In summary, this thesis demonstrates that MYCN can generate various types of brain tumors depending on the timing and location of its expression. It further defines the existence of a rare population of SOX9-expressing MB cells that are involved in causing distant MB recurrences. Finally, it describes how SOX9 is stabilized in MB cells and increases MB migration and therapy resistance.

Le neuroblastome (NB) est un cancer pédiatrique dérivé de la crête neurale. Les NB à haut risque sont des tumeurs peu différenciées présentant une amplification de MYCN et les plus agressives possèdent en plus une mutation d'ALK. Pour améliorer le traitement de ces tumeurs, les nouvelles thérapies cherchent à induire la différenciation cellulaire, l'inhibition de MYCN et la réduction de la signalisation d'ALK. Les résultats obtenus indiquent que le VIP induit une neuritogenèse dans les cellules de NB à haut risque SK-N-DZ et Kelly, et réduit en plus l'expression de MYCN dans les cellules Kelly, comme précédemment observé pour les cellules IMR-32. En parallèle, le VIP diminue l'invasion des cellules Kelly et IMR-32 et réduit également l'activité d'AKT qui est impliquée dans la signalisation d'ALK, dans les cellules Kelly qui présentent la mutation ALK F1174L. Certains effets du VIP sont dépendants de la PKA. Des analogues du PACAP, un peptide apparenté au VIP, présentent une efficacité supérieure à celle du VIP dans les cellules Kelly. Les effets du VIP sur la neuritogenèse et l'expression de MYCN dans ces cellules sont médiés par le récepteur VPAC2 qui peut avoir une localisation nucléaire dans les lignées cellulaires et les cellules de patients atteints de NB. Une délocalisation de ce récepteur nucléaire par ses propres ligands est observée. De plus, des cellules souches mésenchymateuses humaines dérivées du tissu adipeux induisent la différenciation des cellules de NB via les peptides VIP et/ou PACAP. L'ensemble de ces résultats indiquent que le VIP et des analogues du PACAP agissent sur des processus moléculaires et cellulaires qui pourraient réduire l'agressivité des NB à haut risque, et pourraient donc présenter un intérêt pour une nouvelle thérapie. / Neuroblastoma (NB) is a pediatric cancer derived from neural crest. High-risk NB are poorly differentiated tumors with MYCN amplification and the most aggressive forms in addition have an ALK mutation. To improve the treatment of these tumors, the new therapies aim to induce cell differentiation, inhibition of MYCN and reduction of ALK signaling. The obtained results indicate that the neuropeptide VIP induces neuritogenesis in high-risk SK-N-DZ and Kelly NB cells, and in addition reduces the expression of MYCN in Kelly cells, as previously observed in IMR-32 cells. In parallel, VIP decreases Kelly and IMR-32 cell invasion and also reduces the activity of AKT that is involved in the signaling of ALK, in Kelly cells harboring the mutation ALK F1174L. Most of these VIP effects are PKA-dependent. Analogs of PACAP, a VIP-related peptide, exhibit a higher efficiency than VIP in Kelly cells. VIP effects on neuritogenesis and MYCN expression in these cells are mediated by the VPAC2 receptor which can have a nuclear localization in the NB cell lines and in NB from patients. A relocation of this nuclear receptor by its own ligand is observed. Moreover, human mesenchymal stem cells derived from adipose tissue induce NB cells differentiation via VIP and/or PACAP peptides. Taken together, these results indicate that VIP and PACAP analogs act on molecular and cellular processes that might reduce aggressiveness of high-risk NB, and thus could be of interest for new therapy.

Das Neuroblastom (NB) zählt zu den therapieresistentesten Tumoren des Kleinkindalters. Besonders NB-Patienten mit MYCN-Amplifikation sind mit einer schlechten Prognose konfrontiert. Neuere Studien legen nahe, dass eine spezifische aktive Immuntherapie gegen MYCN ein vielversprechender Ansatz zur Bekämpfung dieser malignen Erkrankung darstellen könnte. Zur Untersuchung dieser Hypothese wurde ein sogenanntes Minigen-Vakzin, welches für drei ausgewählte Epitope aus der MYCN-AS-Sequenz kodiert, generiert. Die Auswahl der Minigen-Epitope erfolgte mit dem MHC-Liganden-Vorhersageprogramm syfpeithi, welches drei starke H2-Kk-Liganden lieferte. Außerdem wurden zwei weitere Vakzine hergestellt: als Negativkontrolle MYCNLow, dessen MYCN-Peptide laut syfpeithi schlechte MHC-Klasse-I-Liganden darstellen und ein cDNA-Vakzin auf Basis der gesamten MYCN-cDNA-Sequenz. Zur Applikation der Vakzine wurden attenuierte Salmonella typhimurium SL7207 verwendet, die eine zusätzliche Stimulierung des mukosalen Immunsystems hervorrufen. Für die Untersuchung der Impfstoffe in vivo wurden zwei neuartige immunkompetente NB-Mausmodelle etabliert. Dazu wurden die syngenen NXS2/C1300 A/J-Mausmodelle soweit modifiziert, dass sie über eine induzierbare MYCN-Expression verfügten. Nach Auswahl und Charakterisierung geeigneter Transfektanten in vitro und in vivo wurde in diesen Modellen die Wirksamkeit der Vakzine untersucht. In diesen Versuchen konnte mit Hilfe der Vakzine das Primärtumorwachstum von NXS2-MYCN in geimpften Tieren signifikant im Vergleich zu Kontrollen reduziert und die Metastasierung durch C1300-MYCN Zellen signifikant verzögert werden. Mit den in-vivo-Versuchen anschließenden Analysen wurde schließlich bewiesen, dass die Immunantwort durch tumorinfiltrierende MYCN-spezifische zytotoxische CD8+ T-Zellen vermittelt wird. Zusammenfassend konnte gezeigt werden, dass eine MYCN-DNA-Vakzinierung erfolgreich zur Behandlungen MYCN-exprimierender NB im Mausmodell eingesetzt werden kann. / High-level expression of MYCN protein caused by amplification of the gene characterizes a malignant phenotype of neuroblastoma (NB). Recent studies suggest that MYCN might be a promising target for immunotherapy. Therefore, we investigated the efficacy of three MYCN-specific DNA vaccines. Two minigene vaccines were generated; each encoded for three selected epitopes of the MYCN protein sequence with the highest, or as a control lowest, predicted affinity to MHC-Class-I Molecules. The third vaccine based on the cDNA of MYCN. Salmonella typhimurium SL7207 were used as oral application vehicle for the vaccines in in vivo experiments to induce an additional stimulation of the immune system. To investigate the immunotherapeutic approach NXS2- and C1300-cells syngeneic to immunocompetent A/J-mice were stably transfected with a tetracycline inducible vector system coding for MYCN. The transfectants were characterized and established in vitro and in vivo. In the MYCN-overexpressing models vaccination with the MYCN-DNA-vaccines resulted in significant reduced primary tumor growth or decelerated metastasis spread. The immune responses in the in vivo experiments followed by orally applied MYCN-DNA vaccines was mediated by tumor infiltrating cytotoxic CD8+ and CD4+ T cells. MYCN specificity of infiltrating lymphocytes was verified by MYCN-specific cytolytic activity and IFN-gamma secretion ex vivo. Finally, we showed that blocking of MHC-class I molecule H2-Kk approbated cytotoxicity mediated by CD8+ T cells, indicating MYCN specificity of the induced immune response. In summary, we showed that a MYCN based DNA vaccination strategy is effective against MYCN-expressing NB in vivo. In light of the description of MYCN-specific T cells in NB patients, the lytic action of autologous T cells on MYCN-expressing cells and the results of this study underline the possible potential of an active immunotherapy against MYCN as an alternative therapeutic approach to treat NB.